Cytomechanical Perturbations during Low-Intensity Ultrasound Pulsing

To establish the therapeutic potential of low-intensity ultrasound, it is important to characterize its biophysical interactions with living cells. Here, through a series of single-cell direct observations, we show that low-intensity ultrasound pulsing would give rise to a dynamic course of cytomechanical perturbations at both the membrane and nucleus levels. Our investigation was conducted using a composite platform that coupled a 1-MHz ultrasound exposure hardware to a confocal microscopy system. Short ultrasound pulses (5 cycles, 2-kHz pulse repetition frequency) with a spatial-peak time-averaged intensity of 0.24 W/cm2 (0.85-MPa peak positive acoustic pressure) were delivered over a 10-min period to adherent Neuro-2a neuroblastoma cells, and live imaging of cellular dynamics was performed before, during and after the exposure period. Bright-field imaging results revealed progressive shrinkage of cellular cross-sectional area (25%-45%, N = 7) during low-intensity ultrasound pulsing; the initial rate of size decrease was estimated to be 8%-14% per minute. This shrinkage was found to be transient, as the sonicated cells had recovered (at a rate of size increase of 0.4%-0.9% per minute) to their pre-exposure size within 30 min after the end of exposure. Three-dimensional confocal imaging results further revealed that (i) ultrasound-induced membrane contraction was volumetric in nature (21%-45% reduction), and (ii) a concomitant decrease in nucleus volume was evident (12%-25% reduction). Together, these findings indicate that low-intensity ultrasound pulsing, if applied on the order of minutes, would reversibly perturb the physical and subcellular structures of living cells.